Interaction and uptake of exosomes by ovarian cancer cells

Abstract

BackgroundExosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells.MethodsSKOV3 exosomes were labelled with carboxyfluoresceine diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts.ResultsIn this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-lactose reduced exosomes uptake to a comparable extent as the control D-glucose.ConclusionsIn conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.

Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

“…Furthermore, as the tested conditions could correspond to a high parasite burden present in severe infections, titration of the administered EV dosages and inclusion of different endocytosis-inhibitors could give clues about the specificity and mechanisms of T. muris EV–host interactions. In line with the observation of different studies, uptake of EVs was dramatically reduced when incubating at 4°C, suggesting that internalization is not a passive process occurring in metabolically inactive cells and hence relies on some source of energy [57–60]. A disadvantage of the intestinal organoid culture is the lack of any immune cells.…”

Identification of a novel motif that affects the conformation and activity of the MARCH1 E3 ubiquitin ligase

“…This post-translational modification is known to modulate the incorporation of many proteins into exosomes (Buschow et al, 2005). There is an increasing number of studies reporting the inter-cellular transport of material via exosomes (Simons and Raposo, 2009;Mathivanan et al, 2010;Escrevente et al, 2011). If MARCH1 from IL-10-responsive DCs can be transferred in exosomes to regulate the cell surface display of specific targets in distant APCs remains to be determined (Quah and O'Neill, 2005).…”

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking

“…The initial step is binding of exosomes to the target cell surface through ligand-receptor recognition and lipid-mediated interaction [14,35]. Numerous membrane-associated proteins are sorted into exosomal membranes and are functionally active on target cells [15][16][17][18] and exosome binding occurs within minutes [36,37]. In the second step the attached exosomes are internalized by endocytosis, depending upon the recipient cell type [38].…”